The cDNA of an i type lysozyme was cloned from Stichopus japonicus (named as SjLys). The DNA fragment of the mature SjLys was subcloned into expression vector of pET-32a (+) to construct the recombinant plasmid of pET32a (+)-SjLys. The recombinant plasmid was then transformed into Escherichia coli BL21 (DE3) pLysS and induced by isopropylthio-β-D-galactoside (IPTG). The recombinant protein expressed as inclusion bodies was denatured, partially purified and refolded to be an active form. The bacteriolytic activity of recombinant protein purified by the metal-chelating was 19.2 U/mg. The antibacterial activity of the purified recombinant SjLys (rSjLys) was analyzed. The rSjLys protein displayed inhibitive effect on the growth of the tested Gram-positive and Gram-negative bacteria. In particular, rSjLys had a strong inhibitive activity on Vibrio parahaemolyticus and Pseudomonas aeruginosa, both the most common pathogenic bacteria in the marine animals. The heat-treated rSjLys exhibited more potent activities against all tested bacteria. These results indicated that the S. japonicus lysozyme was the enzyme with combined enzymatic (glycosidase) and non-enzymatic antibacterial action, and it had a wide antibacterial spectrum. Therefore, it is suggested that the S. japonicus lysozyme should be one of the important molecules against pathogens in the innate immunity of sea cucumbers.